GDF15 promotes simultaneous astrocyte remodeling and tight junction strengthening at the blood-brain barrier.

Perivascular astrocyte processes (PAP) surround cerebral endothelial cells (ECs) and modulate the strengthening of tight junctions to influence blood-brain barrier (BBB) permeability. Morphologically altered astrocytes may affect barrier properties and trigger the onset of brain pathologies. However, astrocyte-dependent mediators of these events remain poorly studied. Here, we show a pharmacologically driven elevated expression and release of growth/differentiation factor 15 (GDF15) in rat primary astrocytes and cerebral PAP. GDF15 has been shown to possess trophic properties for motor neurons, prompting us to hypothesize similar effects on astrocytes. Indeed, its increased expression and release occurred simultaneously to morphological changes of astrocytes in vitro and PAP, suggesting modulatory effects of GDF15 on these cells, but also neighboring EC. Administration of recombinant GDF15 was sufficient to promote astrocyte remodeling and enhance barrier properties between ECs in vitro, whereas its pharmacogenetic abrogation prevented these effects. We validated our findings in male high anxiety-related behavior rats, an animal model of depressive-like behavior, with shrunk PAP associated with reduced expression of the junctional protein claudin-5, which were both restored by a pharmacologically induced increase in GDF15 expression. Thus, we identified GDF15 as an astrocyte-derived trigger of astrocyte process remodeling linked to enhanced tight junction strengthening at the BBB.

The Blood-Brain Barrier and the EphR/Ephrin System: Perspectives on a Link Between Neurovascular and Neuropsychiatric Disorders.

Interactions among endothelial cells (EC) forming blood vessels and their surrounding cell types are essential to establish the blood-brain barrier (BBB), an integral part of the neurovascular unit (NVU). Research on the NVU has recently seen a renaissance to especially understand the neurobiology of vascular and brain pathologies and their frequently occurring comorbidities. Diverse signaling molecules activated in the near proximity of blood vessels trigger paracellular pathways which regulate the formation and stabilization of tight junctions (TJ) between EC and thereby influence BBB permeability. Among regulatory molecules, the erythropoietin-producing-hepatocellular carcinoma receptors (EphR) and their Eph receptor-interacting signals (ephrins) play a pivotal role in EC differentiation, angiogenesis and BBB integrity. Multiple EphR-ligand interactions between EC and other cell types influence different aspects of angiogenesis and BBB formation. Such interactions additionally control BBB sealing properties and thus the penetration of substances into the brain parenchyma. Thus, they play critical roles in the healthy brain and during the pathogenesis of brain disorders. In this mini-review article, we aim at integrating the constantly growing literature about the functional roles of the EphR/ephrin system for the development of the vascular system and the BBB and in the pathogenesis of neurovascular and neuropsychiatric disorders. We suggest the hypothesis that a disrupted EphR/ephrin signaling at the BBB might represent an underappreciated molecular hub of disease comorbidity. Finally, we propose the possibility that the EphR/ephrin system bears the potential of becoming a novel target for the development of alternative therapeutic treatments, focusing on such comorbidities.

N-desalkylquetiapine activates ERK1/2 to induce GDNF release in C6 glioma cells: a putative cellular mechanism for quetiapine as antidepressant.

Quetiapine is an atypical antipsychotic which has been suggested to possess also antidepressant efficacy in the treatment of bipolar and unipolar depression. Recently, a link between the activation of the ERK/MAPK signalling pathway and the release of GDNF has been proposed as a specific feature of antidepressants. To obtain a first insight into the putative molecular mechanism of action of quetiapine, we examined its impact and that of its major metabolite norquetiapine on the activation of the ERK/MAPK signalling pathway in C6 glioma cells. Additionally, we investigated the induction of GDNF release as a possible physiological consequence of this activation. We found that norquetiapine, similarly to the antidepressant reboxetine, activated both ERK1 and ERK2 (pERK) with consequent enhanced release of GDNF; this release was dependent on pERK, as demonstrated by its reversibility after pre-treatment with a pharmacological pERK inhibitor. In contrast, quetiapine induced activation of ERK2 only. It also caused release of GDNF, but this release was independent of ERK activation. To test whether the simultaneous activation of ERK1 with ERK2 was critical for the observed pERK-dependent GDNF release, we specifically inactivated ERK1 mRNA via RNA interference. Our data show that indeed ERK1 plays an essential role, as GDNF release was hampered after Erk1 downregulation comparably to a pharmacological pERK inhibitor. Thus, activation of only ERK2 appears not to be sufficient for promoting GDNF release. Our results reveal the release of GDNF as a consequence of ERK/MAPK signalling activation by norquetiapine, which may contribute to the putative antidepressant properties of quetiapine. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Impact of lipid raft integrity on 5-HT3 receptor function and its modulation by antidepressants.

Because of the biochemical colocalization of the 5-HT(3) receptor and antidepressants within raft-like domains and their antagonistic effects at this ligand-gated ion channel, we investigated the impact of lipid raft integrity for 5-HT(3) receptor function and its modulation by antidepressants. Treatment with methyl-beta-cyclodextrine (MbetaCD) markedly reduced membrane cholesterol levels and caused a more diffuse membrane distribution of the lipid raft marker protein flotillin-1 indicating lipid raft impairment. Both amplitude and charge of serotonin evoked cation currents were diminished following cholesterol depletion by either MbetaCD or simvastatin (Sim), whereas the functional antagonistic properties of the antidepressants desipramine (DMI) and fluoxetine (Fluox) at the 5-HT(3) receptor were retained. Although both the 5-HT(3) receptor and flotillin-1 were predominantly found in raft-like domains in western blots following sucrose density gradient centrifugation, immunocytochemistry revealed only a coincidental degree of colocalization of these two proteins. These findings and the persistence of the antagonistic effects of DMI and Fluox against 5-HT(3) receptors after lipid raft impairment indicate that their modulatory effects are likely mediated through non-raft 5-HT(3) receptors, which are not sufficiently detected by means of sucrose density gradient centrifugation. In conclusion, lipid raft integrity appears to be important for 5-HT(3) receptor function in general, whereas it is not a prerequisite for the antagonistic properties of antidepressants such as DMI and Fluox at this ligand-gated ion channel.

Local knockdown of ERK2 in the adult mouse brain via adeno-associated virus-mediated RNA interference.

In recent years RNA interference (RNAi) has become a useful genetic tool to downregulate candidate disease genes for which pharmaceutical inhibitors are not available. In combination with viral vectors to trigger RNAi in the mammalian body, it allows the localized and specific manipulation of the expression of single or multiple genes in vivo. The MAP kinases ERK1 and ERK2 are involved in the transduction of extracellular signals to nuclear effectors. A role for ERKs has been proposed in the adult brain in mediating neuronal functions, as for fear learning in the lateral amygdala. To study the role of ERK in anxiety disorders characterized by disturbed fear learning processes we developed Erk-specific RNAi tools and tested the efficacy of a viral Erk2 vector in the adult mouse brain. We found shRNAs that showed silencing of either both ERK1/2 or only ERK2. In particular, our analysis showed that an Erk2-specific shRNA reduced the activity of this gene at comparable efficiency both in vitro and in vivo. This reagent provides a useful tool to study the role of ERK2, for which small molecule inhibitors are not available, in the development of anxiety and other psychiatric disorders.

Differential mRNA distribution of components of the ERK/MAPK signalling cascade in the adult mouse brain.

The mitogen-activated protein kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs), are a group of serine/threonine terminal protein kinases activated downstream of a pleiotrophy of transmembrane receptors. Main intracellular components of the MAPK signalling pathway are the RAF, MEK, and ERK proteins, which work in a cascade of activator and effector proteins. They regulate many fundamental cellular functions, including cell proliferation, cell survival, and cell differentiation by transducing extracellular signals to cytoplasmic and nuclear effectors. To reveal more details about possible activation cascades in this pathway, the present study gives a complete description of the differential expression of Braf, Mek1, Mek2, Mek5, Erk1, Erk2, Erk3, and Erk5 in the adult murine brain by way of in situ hybridization analysis. In this study, we found that each gene is widely expressed in the whole brain, except for Mek2, but each displays a very distinct expression pattern, leading to distinct interactions of the MAPK components within different regions. Most notably we found that 1) Braf and Erk3 are coexpressed in the hippocampus proper, confirming a possible functional interaction; 2) in most forebrain areas, Mek5 and Erk5 are coexpressed; and 3) in the neurogenic regions of the brain, namely, the olfactory bulb and the dentate gyrus, Braf is absent, indicating that other activator proteins have to take over its function. Despite these differences, our results show widespread coexpression of the pathway components, thereby confirming the hypothesis of redundant functions among several MEK and ERK proteins in some regions of the brain.

Overexpression of p27BBP in head and neck carcinomas and their lymph node metastases.

BACKGROUND: p27(BBP) is a regulator of ribosome assembly and an essential nuclear and cytoplasmic component of eukaryotes. METHODS: We investigated the immunochemical distribution of p27(BBP) in head and neck carcinomas, in the associated normal mucosa, and in regional lymph nodes. RESULTS: p27(BBP) is detectable in mucosal cells but is overexpressed in carcinomas, highly concentrated in large polymorphous nucleoli, and even larger and more evident in lymph node metastatic foci. Western blotting confirms increased p27(BBP) in carcinomas versus normal mucosa and also in metastatic versus normal lymph nodes. The overexpression of p27(BBP) corresponds to mRNA upregulation in carcinomas. Unexpectedly, a 52-kDa band specifically reacting with antibodies to p27(BBP) was observed in several carcinomas. CONCLUSIONS: p27(BBP) alterations are common events in the transition to malignancy and are probably involved in squamous carcinoma progression. Immune reagents raised to p27(BBP) may provide additional diagnostic tools for surgical pathology of tumor boundaries and lymph nodes. The 52-kDa band may represent an abnormal form of p27(BBP) expressed by transformed airway epithelia.

Conversion of cerebral cortex into basal ganglia in Emx2(-/-) Pax6(Sey/Sey) double-mutant mice.

The molecular mechanisms that activate morphogenesis of cerebral cortex are currently the subject of intensive experimental analysis. Transcription factor genes of the homeobox, basic helix-loop-helix (bHLH) and zinc-finger families have recently been shown to have essential roles in this process. However, the actual selector genes activating corticogenesis have not yet been identified. Here we show that high-level expression of at least one functional allele of either of the homeobox genes Emx2 or Pax6 in the dorsal telencephalon is necessary and sufficient to stably activate morphogenesis of cerebral cortex and to repress that of adjacent structures, such as striatum.

Emx2 and Pax6 control regionalization of the pre-neuronogenic cortical primordium.

It has recently been demonstrated that the transcription factor genes Emx2 and Pax6, expressed in the developing cerebral cortex along two complementary tangential gradients, are essential for the shaping of the cortical areal profile at late developmental ages, when cortical neuronogenesis is almost completed. In this study we addressed the question of whether cortical regionalization is already affected in Emx2 and Pax6 loss of function mutants at the beginning of neuronogenesis. By comparing expression patterns of selected molecular markers in these mutants at this age, we found that: (i) Emx2 and Pax6 are necessary for the establishment of their own specific expression profiles and are able to down-regulate each other; and (ii) absence of functional EMX2 or PAX6 proteins results in reduction of caudal-medial and rostral-lateral cortical regions, respectively, as well as in impairment of the WNT signalling center at the medial-caudal edge of the cortical field, crucial for cortical growth. These results suggest that pre-neuronogenic cortical regionalization may rely on mutual interactions between these two transcription factors and that the late areal phenotype of Emx2(-/-) and Pax6(-/-) mutants may possibly arise from both misconfiguration of the cortical molecular protomap and distortion of the cortical growth profile.